Processing blood

ABSTRACT

Methods ( 300 ), devices, and systems of processing blood are described. The method ( 300 ) comprises the steps of: obtaining ( 312 ) blood from a patient coupled to a single blood processing device to form a closed loop between the patient and the blood processing device; collecting ( 314 ) bulk mononuclear blood cells from the blood by leukapheresis implemented using the blood processing device in the closed loop; and enriching ( 316 ) concurrently target cells separated from non-target cells in the bulk mononuclear blood cells using the blood processing device in the closed loop.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit priority to U.S. Provisional ApplicationNo. 61/140,196 filed Dec. 23, 2008, the contents of which areincorporated by reference herein in their entirety.

TECHNICAL FIELD

The present invention generally relates to methods and apparatuses forprocessing blood and more particularly to methods and devices forleukapheresis.

BACKGROUND

Blood cells are produced continuously over the life of an individual andderive from the most primitive blood cell, the so-called hematopoieticstem cell (HSC). This HSC is able to give rise to hematopoieticprogenitor cells (HPC) and to blood cells of the various cell types (egred blood cells (RBC) and leukocytes or white blood cells (WBC)) andtends to be found in the bone marrow. The more mature blood cell typesare found in the blood and lymphatic tissue. Hematopoiesis is thecontinuous production of blood cells in the individual from HSC and HPC.This results in the peripheral blood having many different types ofblood cells of the various myeloid and lymphoid lineages and of varyingdegrees of maturity. These blood cells are responsible for physiologicalprocesses such as oxygen transport by red blood cells, immune functionby dendritic cells, B and T lymphocytes, and inflammatory response bygranulocytes and macrophages.

Apheresis is a medical procedure in which the blood of an individual ispassed through an apparatus, yielding a predominant constituent (e.g.mononuclear cells), and returning the other constituents to thecirculation. Apheresis is in general a three-step process comprising:(1) withdrawing blood from the individual, (2) separating the bloodcomponents (e.g. based on density), and (3) returning certaincomponent(s) of the blood to the individual. The blood is normallyseparated into three fractions: RBC (about 45% of total blood), “buffycoat’ (less than 1% of total blood) and plasma (about 55% of totalblood). Various types of apheresis procedures can be used depending onthe component of blood that is being removed. For example,“plasmapheresis” generally refers to the separation and collection ofblood plasma and “thrombocytapheresis” refers to the separation andcollection of platelets, while “leukapheresis” usually refers to theseparation and collection of leukocytes (WBC).

With the advance of medical sciences, apheresis can be carried out in apatient-connected, closed-loop continuous-flow manner. Devices used forthis purpose include, for example, the following apheresis systems:COBE® Spectra, Trima, Spectra Optia systems (all marketed by Gambro BCT)and the Amicus and CS-3000+ (marketed by Fenwal/Baxter).

Recently, leukapheresis is also being utilised to collect a certainfraction of blood mononuclear cells (MNC) for use in bone marrowtransplantation and other disease areas. For example, patients who havebeen ablated to treat a malignancy can be infused with a bulk populationof donor mononuclear cells that contain HSC and HPC (those present inperipheral blood, also being referred as peripheral blood progenitorcells, or PBPC), to effect subsequent reconstitution of theirhematopoietic system. In this instance, the buffy coat (containing themajority of the WBC (granulocytes, lymphocytes, monocytes), PBPC andsome platelets) is first collected while the remaining components ofblood (including plasma, RBC, platelets and some WBC) are returned tothe individual. The PBPC are then enriched and isolated, while theremaining fraction of the buffy coat (constituting nearly 99% of thebuffy coat) is discarded. This process of cell enrichment (i.e. cellisolation and purification) is currently carried out in apatient-disconnected manner, using separate devices to those of theapheresis machines. Devices used for this purpose include, for example,the Baxter Isolex 300i and the Miltenyi CliniMACS, which enrich PBPCbased on a specific ligand (CD34, both devices and CD133 Miltenyi) onthe cells' surface. Other stand-alone devices, such as the Gambro COBE2991 Blood Cell Processor or the Baxter CytoMate™ Cell Washing System isoften used to wash, concentrate, or place the cells into appropriategrowth or infusion medium.

In a further application, leukapheresis can be used to treat anindividual's WBC in a process called photopheresis (Edelson et al., YaleJ Biol Med. 1989 November-December; 62(6): 565-77). In this process, theindividual first receives a dose of photoactivatable substance (e.g. 8methoxy-psoralen). Then apheresis is carried out in which the WBC of theindividual is irradiated with Ultraviolet A (UVA) light, resulting inthe activation of the substance and inhibition of the metabolicprocesses of the WBC. Devices used for this purpose include, forexample, the UVAR AND UVAR® XTS™ Photopheresis System (marketed byTherakos).

In addition to the enrichment process described above, the PBPCcollected may also be modified in further processes before re-infusingback to the individual. This is generally effected by the use of avariety of techniques in cell culture. Ultimately, the modified cells(for example, altered phenotype, genotype or activity) may bere-introduced into the patient for certain therapeutic benefits.Examples of modification processes include the production of HSC/HPCcontaining an anti-HIV gene (R. G. Amado. et al. Human Gene Therapy 15(2004), 251-262) and the production of cytotoxic T lymphocytes‘educated’ to home to and kill specific tumours.

FIG. 1 is a block diagram illustrating the current means by which bloodis removed from a patient, processed and returned. Here, an arrangement100 of devices that can be sequentially used for cell collectionemploying leukapheresis and cell enrichment techniques, such as usingcell washing, purification. The diagram also lists optional cellmodifications. Exemplary devices that can be used in this method includethe Cobe Spectra device. Such a device, 110, for leukapheresis(collection), is used sequentially with a cell-washing device, such as aBaxter CytoMate device, 120, (enrichment) which is further used with acell purification (enrichment) device such as a Baxter Isolex 300idevice, 130. In addition, cell manipulation (modification) devices, 140,can be employed in this scheme and include, but are not limited to:electroporation, lipofection, viral transduction, light (UVA, UVB,etc.), addition of drugs, cell activation, pressure, heating functions,etc. The bag 150 of processed blood cells produced as the output ofdevices 110, 120, 130, and 140 are provided to the patient 160 forreturn of the processed blood.

FIG. 2 depicts a specific example of a method 300 for cell collection,enrichment and modification. The example is of a method used for theintroduction of an anti-HIV gene into CD34+ HSC/HPC in which over a 5day period:

In step 310, mononuclear cells are collected, i.e. harvested byleukapheresis. In this step 310, other blood cell components, namely redblood cells, platelets, plasma and polymorphonuclear cells are returnedto the patient.

In step 320, the mononuclear cell fraction is washed using, for example,a CytoMate (referenced above) (day 2), target CD34+ cells are enrichedusing, for example, an Isolex 300i device (day 2), and non-CD34+ cellsare discarded.

In step 330, the CD34+ cells are cultured in the presence of cytokines(day 2), and the anti-HIV gene (a ribozyme against a conserved region ofthe tat/vpr gene) is introduced using a murine retrovirus (day 4).

After step 330, the product release testing is performed (day 5), andthe cells are infused to the same individual, who was originallyleukapheresed.

However, apheresis has inherent drawbacks and limitations. For example,apheresis is only a fluid constituent(s) collection procedure. Despitetechnological advances, the composite steps of collection, enrichmentand (optional) modification of target blood cells are conducted by usingseparate continuous and discontinuous devices, as mentionedhereinbefore. Of these steps, only collection and in one instance,collection and modification (photopheresis) are currentlypatient-connected. These current discontinuous processes are timeconsuming and materials, labor and costs inefficient (J. Gryn et al.,Journal of Hematotherapy & Stem Cell Research 11 (2002), 719-730; K. R.Meehan et al., Journal of Hematotherapy & Stem Cell Research 9 (2000),767-771). These processes also introduce serious concerns such as (i)safety due to potential microbial contamination and (ii) chain ofcustody (i.e. ensuring the correct cells are returned to the patient andmaintaining the cells' integrity) due to the logistics of cell selectionand modification. For instance, hemolysis is a rare complication due tokinks in the lines of the apheresis collection kits (R. Reddy,Transfusion and Apheresis Science 32 (2005) 63-72).

To further illustrate, thrombocytopenia (depletion of platelets) is awell-known unwanted result of leukapheresis and the most frequentlyreported secondary effect of leukapheresis in children (J. Sevilla etal., Transfusion and Apheresis Science 31 (2004) 221-231; E. Yamaguchiet al., Journal of Hematotherapy & Stem Cell Research 9 (2000) 565-572).Thrombocytopenia is important, because patients are oftenthrombocytopenic due to their underlying diseases and there is anadditional loss of platelets during leukapheresis. Ideally, inindividuals with a deficiency in platelet numbers due to certain diseasestates, the apheresed platelets within the buffy coat should beseparated and returned to the individual. In reality, however, theapheresed platelets are simply discarded as wastes. This aside, thereduction of platelets in the buffy coat also has the added benefit ofincreasing the efficiency of immunoaffinity selection of CD34+progenitor cells (a type of PBPC) by a mean of 1.8 fold (R. Moog,Transfusion and Apheresis Science 31 (2004) 207-220).

Another drawback of the leukapheresis process is the loss of valuablelymphocytes for some patients. As discussed hereinbefore, HSC and HPC(in particular CD34+ progenitor cells) are often selected for use toeffect reconstitution of an individual's hematopoietic system. For humanimmunodeficiency virus (HIV) infected individuals, the selection ofCD34+ progenitor cells using leukapheresis rid their body of valuablelymphocytes (such as CD3+ and CD4+ cells), which are often already lowin numbers. CD34+ progenitor cells—around 1.3% after mobilization—areamong the smallest cell fraction collected during PBPC leukapheresiswhereas lymphocytes and monocytes account for up to 70% of the apheresisproducts (V. Witt et at., Journal of Clinical Apheresis 16 (2001)161-168).

A need exists for a device that can overcome or at least ameliorate oneor more disadvantages of existing systems, including those mentionedhereinbefore.

SUMMARY

In accordance with an aspect of the invention, there is provided anapparatus for processing blood. The apparatus comprises: an inletinterface for coupling with a patient to receive blood directly from thecirculation of the patient; a leukapheresis module coupled to the inletinterface for collecting bulk mononuclear blood cells from the receivedblood; an enrichment module coupled to the leukapheresis module forenriching concurrently target cells separated from non-target cells inthe bulk mononuclear blood cells; an outlet interface coupled to atleast one of the leukapheresis module and the enrichment module forcoupling with the patient to return enriched target cells to thecirculation of the patient, the apparatus and the patient forming aclosed loop when coupled together; and a controller for automatedcontrol of operation of the inlet and outlet interfaces, theleukapheresis module, and the enrichment module.

In accordance with another aspect of the invention, there is provided amethod of processing blood. The method comprises the steps of: obtainingblood from a patient coupled to a single blood processing device to forma closed loop between the patient and the blood processing device;collecting bulk mononuclear blood cells from the blood by leukapheresisimplemented using the blood processing device in the closed loop; andenriching concurrently target cells separated from non-target cells inthe bulk mononuclear blood cells using the blood processing device inthe closed loop.

In accordance with a further aspect of the invention, there is provideda system for processing blood. The system comprises: a mechanism forobtaining blood from a patient and comprises a single blood processingdevice coupled to the obtaining means and the patient to form a closedloop between the patient and the blood processing device. The bloodprocessing device comprises: a module for collecting bulk mononuclearblood cells from the blood by leukapheresis implemented using the bloodprocessing device in the closed loop; and a module for enrichingconcurrently target cells separated from non-target cells in the bulkmononuclear blood cells using the blood processing device in the closedloop.

These and other aspects of the invention are set forth in greater detailhereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are described hereinafter with reference tothe drawings, in which:

FIG. 1 is a block diagram of devices for cell collection byleukapheresis and enrichment using cell washing and purification;

FIG. 2 is a schematic diagram depicting a specific example of a currentmethod for cell collection, enrichment and modification;

FIG. 3 is a flow diagram of a method of processing blood cells inaccordance with an embodiment of the invention;

FIG. 4 is a schematic diagram depicting concurrent cell collection,target cell enrichment and return of non-target cells;

FIG. 5 is a schematic diagram depicting concurrent cell collection,target cell enrichment and return of target cells;

FIG. 6 is a schematic diagram depicting a method of concurrent cellcollection from a patient, enrichment of target cells, modification oftarget cells, and return of modified target cells to the patient;

FIG. 7 is a schematic diagram of a device performing collection andenrichment in accordance with an embodiment of the present invention;Symbols represent the following components: Saline bag,

peristaltic pump,

clamp,

air detector,

pressure gauge,

clamp,

FIG. 8 is a schematic diagram of a device performing collection,enrichment, and modification in accordance with another embodiment ofthe present invention Symbols represent the following components: Salinebag,

peristaltic pump,

; clamp,

; air detector,

pressure gauge,

clamp,

FIG. 9 is a perspective view of elements of the blood-processing devicecomprising the collection, enrichment and (optional) modification unitsor modules;

FIG. 10 is a perspective view of elements of the blood processingdevice, per the device of FIG. 9, but highlighting the cell enrichmentmodule/process;

FIG. 11 is a perspective view of elements of the blood processingdevice, per the device of FIG. 9 or 10, but highlighting the optionalcell modification step within the device; and

FIG. 12 is a schematic diagram of a device performing collection andenrichment in accordance with still another embodiment of the presentinvention. Symbols represent the following components: Saline bag,

peristaltic pump,

clamp,

air detector,

pressure gauge

clamp,

magnet,

DETAILED DESCRIPTION

Methods, apparatuses, and systems for processing blood cells aredescribed hereinafter. In particular, methods, apparatuses, and systemsare disclosed for leukapheresis that enable the concurrent collectionand enrichment of specific target cells from an individual's peripheralblood and the remaining blood components are returned to the individual.Additionally, the target cells collected may be modified and returned tothe individual during the apheresis process, or may be returned to theindividual at a later time. The embodiments of the invention relate to aclosed-loop device that enables the concurrent collection and enrichmentof specific target cells from peripheral blood of an individual andreturn of the non-target cells to the individual. The target cells maybe modified to alter their phenotype, genotype or activity and in anextension of the closed-loop returned to the individual. The embodimentsof the invention efficiently carry out the process of apheresis in apatient-connected, closed-loop continuous-flow manner, whereby onlytarget components of blood (e.g. CD34+ progenitor cells) are enrichedwhile all other remaining components are returned to the patient.Additionally, certain other functions may be carried on the target cells(e.g. modifying the phenotype) with the option of returning the modifiedcells to the patient. The provision of such a device can significantlyreduce operating costs (no need of multiple apparatus and consumables)and ensure product consistency. Enabling the apheresis procedure tooccur in a single location in a single device also reduces the risk ofdamage or loss of the product.

However, from this disclosure, it will be apparent to those skilled inthe art that modifications and/or substitutions may be made withoutdeparting from the scope and spirit of the invention.

DEFINITIONS Term Definition Bulk The bulk mononuclear cell populationcollected by Mononuclear leukapheresis. Also referred to as BulkMononuclear Cell Cell Population. Capture The isolation of one specificcell type or types from a mixture of cells by a specific interaction (egphysical or chemical) (eg antibody-antigen or other interactions asdescribed herein). This may be done by means of a cell capture system.Cell Processing All steps (some of which may be optional) that involvecollecting, enriching, modifying and storing cells. The cells may beused outside the body for research, monitoring or discarded or they maybe subsequently infused to an individual(s). Closed-loop At least asubsection of cells is kept in a system that is a continuous, such thatthe cells are derived from the patient and can be returned to thepatient without being moved off-line. Cluster Classification system formonoclonal antibodies Designation generated by laboratories worldwideagainst cell (CD) surface molecules on leukocytes initially, now alsoantigens from other cell types. Concurrent Part of same real-timeprocess, occurring in real time; it can be sequential or simultaneous.Continuous Part of a closed-loop. Continuous flow The flow of blood fromthe patient to the device and back to the patient in which non-targetcells generally return to the patient whereas target cells may becollected, may flow past an enrichment system in the device, may bemodified by a modification system in the device and then return to thepatient; all in a closed-loop patient-connected manner and in real time.Continuous Flow The fluid path and engineering controls that permitSystem Continuous flow. Differential Centrifugation to separate a bulkpopulation of cells Centrifugation on the basis of size and density.Discontinuous Not part of a closed-loop. Enrich The concentration of thetarget cell type by a physical or chemical means; this may encompasswashing, isolation or purification. Integral An important partof/requirement. Isolate The process of extracting (eg by capture) onespecific cell type or types from a mixture of cells. Leukapheresis Acontinuous flow process where blood is drawn from a donor and a bulkmononuclear cell population is collected and the remaining components ofblood (plasma, platelets, red blood cells and polymorphonuclear cells)are re-infused to the donor. Ligand Any molecule (eg an antibody) thatbinds to another (eg a receptor). Monitor Determination of parameters ofprocess (eg real-time or post-hoc) eg measurement of number of aspecific cell type (eg CD34) being captured. Non-Target Cells These arethe cells remaining after the enrichment of target cells from the bulkmononuclear cell collection, which has been collected by leukapheresis.Modification The alteration of cell phenotype, genotype or activity;also referred to in some instances as manipulation. Patient-connectedThis refers to when the device is connected to the patient. The patientmay be connected to the device for the entire period of blood cellcollection and enrichment and potentially cell modification. The flow ofblood is from the patient to the device and back to the patient in whichnon-target cells generally return to the patient whereas target cellsmay be collected, may flow past an enrichment system in the device, maybe modified by a modification system in the device and then return tothe patient; all in a closed-loop manner. Patient- Steps of cellprocessing that occur when the patient is disconnected disconnected fromthe device. Purity The percentage of a specific cell type in the cellpopulation. Real-Time As it is occurring Real-Time Determination ofparameters of process as it is Monitoring occurring eg measurement ofnumber of a specific cell type (eg CD34) being captured. Release Theprocess of separation (eg physical or chemical) of the cell from thecapture system. Same device One device in which multiple functions maybe performed. Target Cell The cell type or types enriched from the bulkmononuclear cell population collected by leukapheresis. The target celltype (or types) is enriched for subsequent discarding or for subsequentuse which may include modification and re-infusion to the individual. Itis understood that the target blood cell type that is enriched canencompass one or more cell types. Also referred to herein as Target CellType or Target Cell Population.

Multi-functional devices and methods of use thereof, that isclosed-loop, can be patient-connected and comprise the followingaspects:

-   -   a) Collection: performing leukapheresis collection of a bulk        mononuclear blood cell population that contains the target cell        population of interest; and concurrently    -   b) Enrichment: enriching a target cell population from the bulk        mononuclear blood cell population

The enriched target cell population is either returned to the patient,removed for subsequent off-line use including modifications that mayinvolve the later infusion of the modified cells to the patient. Theoff-line use of the target cell population may include use for researchor monitoring. The non-target cell population may be concurrentlyreturned to the patient, removed off-line for use or optionallydiscarded. The method can additionally, in an extension of theclosed-loop process, modify the target blood cell population beforereturning the modified target blood cell population to the patient.

FIG. 3 depicts at a high level a method 300 of processing bloodcomprising steps 310-316 and 330 (indicated by boxes having unbrokenlines). While not depicted in FIG. 3, the steps 312-316 may be carriedout repeatedly. The method 300 may optionally comprise one or more ofsteps 320-326 (indicated by boxes having dashed lines in FIG. 3).Likewise, one or more of these steps 320-326 may be performed repeatedly(not shown in FIG. 3). While the steps of method 300 are depicted asbeing performed sequentially and in a particular order, the method 300is not limited to the particular sequence, sequential processing, or allof the steps, some of which are optional, being performed. It will beunderstood by those skilled in the art that in the light of thisdisclosure the ordering of steps may be changed. Further, one or moresteps may be performed in parallel. For example, steps 312 to 316 may beperformed in parallel. Still further, step 326 may be performed inparallel with steps 314 and 316. The method 300 of processing blood isdescribed in greater detail hereinafter.

Processing commences in step 310. In step 312, blood is obtained from apatient coupled to a single blood-processing device to form a closedloop between the patient and the blood processing device.

In step 314, bulk mononuclear blood cells are collected from the bloodby leukapheresis implemented using the blood-processing device in theclosed loop. The collecting step 314 may comprise using differentialcentrifugation to collect the mononuclear blood cells. The differentialcentrifugation may be conducted by a continuous flow system.

In step 316, target cells separated from non-target cells in the bulkmononuclear blood cells are enriched concurrently using theblood-processing device in the closed loop. The target cells may be Bcells, T cells, dendritic cells, monocytes, neutrophils, natural killer(NK) cells, T regulatory cells, T helper cells, cytotoxic T lymphocytes(CTLs), hematopoietic stem cells (HSCs), hematopoietic progenitor cells,endothelial cells, epithelial cells, mesenchymal cells, lymphocytes,lymphokine activated killer cells (LAKs), or tumor infiltratinglymphocytes (TILs). The T cells may be enriched. The T cells may be CD8+or CD4+. The hematopoietic progenitor cells and the hematopoietic stemcells may be enriched. The hematopoietic stem cells and thehematopoietic progenitor cells may be positive for one or more of CD34,CD133, and CD143. Alternatively, the target cells may be at least one ofmalignant cells from blood, malignant cells from tissue, virallyinfected cells, bacterially infected cells, at least one virus, at leastone bacterium, a parasite, fetal cells, and pathogenic effector cells.

The enriching step 316 may comprise ligand capture to enrich the targetcells. The ligand may be an antibody specific for a cell surface ligand.The cell surface ligand may be an epithelial cell adhesion molecule(EpCAM), a selectin, an adhesion molecule receptor, a homing receptor, acytokine receptor, a chemokine receptor, or an enzyme. The cell surfaceligand may be a cluster designation (CD) antigen. The CD antigen may beCD1a, CD4, CD8, CD14, CD25, CD34, CD133, or CD143. The target cellenrichment in step 316 may be effected by at least one of magnetics,fluorescent activated cell sorting, microfluidics, solid support,acoustics, bioluminescence, antibody tagging, and enzyme substrate. Thesolid support may comprise a particle. The particle may be at least oneof a magnetic particle and a density modified particle.

The collecting and enriching steps 314, 316 may be performed indifferent sections of the blood-processing device.

In step 320, the enriched target cells may be modified. The modifyingstep 320 may involve modification comprising one or more of activation,expansion, induction of apoptosis, gene modification, and induction ofantigen specificity. The modifying step 320 may involve modificationthat is effected by at least one of cross linking cell surfacereceptors, irradiation, and treatment with at least one of cytokines,chemokines, antigen stimulation, hormones, drugs, pressure, and heating.The irradiation may be at least one of gamma, beta, alpha, and lightradiation. The light radiation may be at least one of ultraviolet A(UVA), ultraviolet B (UVB), and visible light. Alternatively, themodifying step 320 may involve genetic modification that is effected byone of transfection and transduction of genetic material into at least aportion of the target cells. Transfection of genetic material may be byone of electroporation and lipofection. Transduction of genetic materialmay be by viral vector transduction. In yet another alternative, themodifying step 320 may involve modification comprising at least one ofcytotoxic T lymphocyte (CTL) activation, T regulatory cell (Treg)activation, and genetically modified blood cells protected from humanimmunodeficiency virus (HIV).

In step 322, non-target cells may be modified. Further, in step 324, thenon-target cells may be returned to the patient. The non-target cellsmay be returned to the patient connected in the closed loop, ordisconnected from the closed loop. Still further, the non-target cellsmay be discarded.

In step 326, the collecting and enriching steps may be monitoredconcurrently for cell number. This would allow the collection to becompleted as soon as sufficient cells have been collected and enriched,allowing the collection to be tailored to the patient.

The method may comprise maintaining continuous connection of the patientin the closed loop during processing of the target cells, ordisconnecting the patient from the closed loop for a time intervalduring processing of the target cells. Processing terminates (End) instep 330. These and other aspects are described in greater detailhereinafter.

Concurrent Cell Collection and Enrichment of Target Cells for Off-LineUse Including Modification or for Discarding

FIG. 4 depicts at a high level a method 400 of concurrent cellcollection 410 from the patient 450, target cell enrichment 420 andreturn of non-target cells 452 (all depicted within circle 402). Theenriched target cells 430 may be used off-line for research/testing 462,470 or discarded 482. Alternatively, the enriched target cells 430, 462are modified for infusion 464 of the target cells to the patient 450 ata later time. This aspect encompasses concurrent cell collection 410,target cell enrichment 420 and return 452 of non-target cells to thepatient 450 in a closed loop. The leukapheresis collection step 410yields a mononuclear cell population. An online enrichment step 420 oftarget cells is performed. The non-target cells are returned 452 to thepatient 450. The enriched target cells 430 may be used off-line and mayoptionally be modified for research, testing etc 470 or infusion 464 ofthe target cells to the patient 450 at a later time. The target cells462 may be used with or without modification. The cells may also bediscarded 482. In the situation where target cells are modified andreturned 464 to the patient, non-target cells 452 may not necessarily begiven back to the patient 450. The process 400 of concurrent cellcollection 410, target cell enrichment 420 and off-line cellmodification may be repeated as many times as necessary, for example, toreach a certain number of target cells. Target cell enrichment 420 maybe for one or several cell types. Target cell modification may be forone or several cell types. The closed loop is connected to the patient450 at the times of cell collection 410 and cell return 452.

Concurrent Cell Collection and Enrichment of Target Cells for Returnwith Non-Target Cells Used Off-Line or Discarded

FIG. 5 depicts at a high level a method 500 of concurrent cellcollection 510 from a patient 550, target cell enrichment 520 and returnof target cells 552 to the patient 550. The enriched non-target cells530 are then used off line with or without modification forresearch/testing 562, 570 or are discarded 582. Again, the leukapheresiscollection step 510 yields a mononuclear cell population. The onlineenrichment step 520 of target cells is performed. The target cells 552are returned to the patient 550. This aspect encompasses concurrent cellcollection 510, enrichment 520 of target cells and return 552 of targetcells to the patient 550. Non-target cells 562 may be used off-line forresearch, testing etc 570 (either with or without a modification step)or are discarded 582. The closed loop is connected to the patient 550 atthe times of cell collection 510 and cell return 552.

Concurrent Cell Collection, Enrichment and Modification of Target Cells

FIG. 6 depicts at a high level a method 600 of concurrent cellcollection 610 from a patient 650, enrichment 620 of target cells,modification 660 of target cells 630, and return of modified targetcells 670 to the patient 650. Non-target cells 652 may also be returnedto the patient 650. This aspect encompasses concurrent cell collection610, enrichment 620 of target cells, modification 660 of target cellsand return of modified target cells 670 to the patient 650 in a closedloop procedure. Non-target cells 652 may optionally be returned to thepatient. The closed loop is connected to the patient 650 at the times ofcell collection 610 and cell return 652, 670.

Cell Collection

An embodiment of the invention comprises cell collection and concurrentcell enrichment. Collection is the leukapheresis collection of the bulkmononuclear blood cells, the cells from which the target blood cells areenriched. This step can employ any method known in the art for obtainingmononuclear cells from a patient including, without limitation, the useof differential centrifugation. Devices for this purpose include theCOBE® Spectra, Trima Spectra Optia systems (all marketed by Gambro BCT)and the Amicus or CS-300 (marketed by Fenwal/Baxter) Gambro Cobe Spectraor Optia, Fenwal Amicus or CS-3000. Preferably, the differentialcentrifugation is conducted by a continuous flow system. In a preferredembodiment, this bulk blood cell collection uses the Therakos CellExtechnology due to its superior collection efficiency and lowextracorporeal volume compared to other devices, which includes suchdevices as listed above. During leukapheresis, the non mononuclear cellpopulation is reinfused to the individual.

FIG. 9 illustrates the blood processing device in accordance with anembodiment of the invention. The patient 940 is coupled to the device900 in a closed loop fashion with an input catheter 950 coupled to thepatient 950 to provide blood as input to the device 900 and an outputcather 952 as a return path from the device 900 to the patient. Thedevice 900 has an inlet interface to receive blood directly from thecirculation of the patient. The device 900 also has an outlet interfaceto return enriched target and/or non-target cells to the circulation ofthe patient. The device 900 and the patient form a closed loop whencoupled together. The device 900 includes a leukapheresis collectionunit 910 and an enrichment unit 920. The (leukapheresis) collection unitor module 910 collects bulk mononuclear blood cells from the receivedblood. The enrichment unit or module 920 enrichs concurrently targetcells separated from non-target cells in the bulk mononuclear bloodcells. The device 900 has an operator interface 960 for receiving inputsand providing outputs to an operator (not shown). The device 900 alsocomprises a pump/valve deck 964. The device 900 may also comprise anoptional modification unit 930. The device 900 comprises a centrifuge962 for processing blood cells as explained hereinafter. A controller(not shown) is coupled to the operator interface 960 and the othermodules for automated control of operation of the device 900 In theTherakos CellEx system, a centrifuge bowl, such as, for example, aLatham bowl, as shown in U.S. Pat. No. 4,303,193 issued to Latham, Jr on1 Dec. 1981 and entitled “Apparatus for separating blood into componentsthereof”, which is incorporated herein by reference in its entirety,separates blood into red blood cells and “buffy coat”. The Latham bowlis a blood component separator that has been used for some time in themedical leukapheresis market as well as in medical therapies such asextracorporeal photopheresis (ECP). U.S. Pat. No. 5,984,887“Photopheresis treatment of leukocyte” provides descriptions ofextracorporeal photopheresis and its method of cell separation andcentrifugation.

FIG. 7 is a more detailed schematic diagram of the blood-processingdevice depicted in FIG. 9 (as well as the system of FIG. 10 describedhereinafter). The blood-processing device 700 is shown coupled to apatient 736 in FIG. 7. A collection node 702 and a return node 756 areconnected to the patient in the manner shown in FIG. 9. The collectionnode 702 is part of an input interface including a catheter 704, whichis in turn connected to a pressure (“collect”) sensor 720. In sequence,the collect pressure sensor 720 is coupled to an air detector 722, whichis coupled to a collect valve 724. The collect valve 724 is coupled to a“collect” peristaltic pump 726, which in turn is coupled to the bowlpressure sensor 728. The pressure sensor 720 affects operation of thecollect pump 726. The bowl pressure sensor 728 is coupled to thecentrifuge bowl 730. One output of the centrifuge bowl 730 is coupled toa red blood cell pump (RBC) 732 and catheter 734, which is in turncoupled to a return path, described in greater detail hereinafter

An anticoagulant (AC) bag 710 is coupled to an anticoagulant peristalticpump 712 and appropriate catheter. The pump 712 is in turn coupled to avalve 714, which in turn is coupled to an air detector 716. The airdetector 716 is coupled by a suitable catheter to the input catheter 704and collect pressure sensor 720. This arrangement allows anticoagulantto be applied to blood input to the device 700 from the patient 736.

Another catheter 770 provides an output from the centrifuge bowl 730 andis coupled to a valve 772. Also coupled to the catheter 770 is acatheter 782 coupled to a valve 784. In turn the valve 784 is coupled toa return bag 740. The return bag 740 is coupled to an air detector 742,which in turn is coupled to a valve 744. The valve 744 is in turncoupled to a valve 798, a saline valve 760, which is in turn coupled toa saline bag 762, catheter 734 and a return pump 746. The return bag740, air detector 742, valve 744 form a return path with the return pump746. The pump 746 is coupled to the return valve 748, which is coupledto an air detector 750. The air detector 750 is coupled to the returnpressure sensor 752, which is coupled to catheter 754 and return node756.

The valve 772 is coupled to a sensor 788 capable of detecting red bloodcells. A catheter 774 is also coupled to the valve 772 and in turn isconnected to a buffy pump 776. The buffy pump 776 is coupled to a plate778. The output of plate 778 is coupled to a catheter 797, which in turnis coupled to valve 798. Valve 798 is coupled to return pump 746. TheHCT sensor 788 is coupled to parallel-configured valves 790 and 791. Thevalve 790 is coupled to a collection bag 786. The valve 791 is coupledto treatment bag 737 where agents for enrichment are added. Thetreatment bag 737 is coupled to valve 793, which in turn is coupled toair detector 794. The air detector 794 is coupled to valve 798.

A selection buffer bag 795 is coupled to a valve 796, which in turn iscoupled to air detector 794.

Cell Enrichment

FIG. 10 shows the device 900 of FIG. 9 renumbered as device 1000. Thepatient 1040 is coupled with the device 1000 in a closed loop fashionwith input catheter 1050 and output catheter 1052. In this embodimentthe mononuclear cell population 1010 is subject to enrichment 1020, e.g.by antibody coated particle capture, such as magnetic particle capture.The output of enrichment 1020 is the enriched target cells 1060, whichcan be returned to the patient. Also remaining non-target cells from theenrichment 1020 may be returned to the patient 1040 via the device 1000.Cells are enriched for specific purposes, which include but are notlimited to:

-   -   1. Elimination from the blood stream (eg leukemic lymphoma,        myeloma cells); these cell types would be selected as cells to        be eliminated from the blood and discarded.    -   2. Modification to give back to the patient for positive        benefit; some examples include:        -   a. eliciting an immune response by enrichment and            modification of leukemic cells or metastatic cancer cells;        -   b. modification to generate cytotoxic T lymphocytes targeted            to a specific cancer; and        -   c. modification of HSC/HPC to contain a gene to impact on a            disease process, eg an anti-HIV gene to impact on HIV/AIDS.    -   3. Use for research or testing etc, which may include an        optional modification step.

Target cells are cells that are enriched from peripheral blood post bulkmononuclear cell collection. Cell types that can be enriched from theleukapheresis bulk product include but are not limited B lymphocytes, Tlymphocytes, CD4 and CD8 T lymphocytes, dendritic cells, monocytes,natural killer (NK) cells, T-regulatory cells, T-helper cells, cytotoxicT lymphocytes (CTLs), hematopoietic stem cells (HSCs), hematopoieticprogenitor cells, endothelial cells, epithelial cells,lymphokine-activated killer cells (LAKs), tumor infiltrating lymphocytes(TILs), mesenchymal stem cells and epithelial cells—see Table 1(Fundamental Immunology By William E. Paul 2003 Lippincott Williams &Wilkins ISBN 0781735149; Essential Haematology, Hoffbrand, Pettit andMoss).

TABLE 1 Cell Types That Can be Isolated From Peripheral Blood (withknown surface markers) Hematopoietic Progenitor Cells (CD34⁺, CD135⁺)Endothelial Progenitor Cells (CD34⁺, Flk-1⁺, VEGRF-3⁺, CD133⁺) BoneMarrow Stromal Cells Skeletal Muscle Progenitor Cells Cardiac MuscleProgenitor Cells Hepatic Progenitor Cells (C1qR_(p) ⁺ or CD34⁺, CD38⁻,CD45⁺) Mesenchymal Stem Cells (CD29⁺, CD44⁺, SH2⁺, SH3⁺, and SH4⁺)Erythrocytes (CD44, Glycophorin A) Dendritic Cells (CD11c⁺, CD123⁺) TCells (CD3⁺, CD4⁺, CD8⁺, CD28⁺) NK Cells (CD16⁺, CD57⁺, CD94⁺, CD96⁺,CD122⁺) B Cells (CD19⁺, CD22⁺, CD40⁺, CD72⁺, CD79⁺) Neutrophils (CD15⁺,CD128⁺) Eosinophils (CD116⁺, CD125⁺) Basophils (CD125⁺) Monocytes -Macrophages (CD14⁺, CD64⁺, CD68⁺, CD98⁺, CD115⁺, CD163⁺, Flt-1⁺)Megakaryocyte/Platelets (CD41⁺, CD42⁺, CD61⁺, CD109⁺) Mast Cells(FcεRIα⁺) Osteoblast Progenitors* Osteoclasts* *Isolated from peripheralblood but no cell surface markers identified to date.

Other cell types targeted for discarding can be any known in the art,including, without limitation cancer/leukemia cells from blood or othertissues, viral or bacterially infected cells, viruses or bacteria orparasites, fetal cells, or pathogenic effector cells. These cells can beenriched by the use of appropriate surface antigens. These latter cellscan also be targeted for modification as per purpose #2 above, iemodification of cells and giving the modified cells back to effect atherepeutic immune response.

During the enrichment step, more than one target cell type may beenriched. The system may enrich multiple cell types in various ways, egthe cell types may be enriched separately in different chambers of thedevice (900, 1000 of FIGS. 9 and 10). The different cell types may bemanaged together (eg all returned or discarded or modified) or the celltypes may be managed separately (eg one set returned, one set discarded,one set modified or all sets modified but in different ways) orvariations of the preceding.

The enrichment of the target cell(s) may be to eliminate the target cellfrom the peripheral blood (as in leukemia cells) or to enrich to apercentage purity required for the therapeutic application or forresearch/testing etc.

Enrichment of the target cell may be by chemical or physical means, egcapture, and the target cells are said for example, to be isolated, thatis enriched from the bulk blood cell population. The enrichmentprocedure may employ one or more methods known in the art including,without limitation, antigen capture, beads, magnetics,fluorescent-activated cell sorting, microfluidics, solid support,acoustics, bioluminescence, antibody tagging, or enzyme substrate.Suitable solid supports include particles including, without limitation,ferromagnetic and density modified particles. These can be obtained, forinstance from Miltenyi Biotec and Dynal (Curr Opin Immunol. 1991 April;3(2):238-241). There exist methods that can be used for the release ofthe captured cells that include: i) competition with excess ligand, ii)enzymatic digestion, iii) change in pH, iv) change in ionic strength, v)removal of magnetic field, vi) physical agitation.

The ligand/s specific for the target cell population or populations canbe any known in the art and is preferably an antibody specific for acell surface ligand. The cell surface ligand can be a clusterdesignation (CD) antigen including, without limitation, CD1a, CD4, CD8,CD14, CD25, CD34 and CD133, which usually utilizes a specific antibodyto capture/select the target cell. The cell surface ligand can be,without limitation EpCAM (epithelial cell adhesion molecule), selectins,adhesion molecule receptor, homing receptors, cytokine receptors,chemokine receptors and enzymes including aldehyde dehydrogenase andother intracellular enzymes. Various surface markers are indicated inTable 1.

As one example, one way of enriching cells is the use of antibodies oraptamers. The term antibody refers to an immunoglobulin molecule capableof binding an epitope present on an antigen. As used herein, the termantibody refers to cell-binding molecules. The term is intended toencompasses not only intact immunoglobulin molecules such as monoclonaland polyclonal antibodies, but also bi-specific antibodies, humanizedantibodies, chimeric antibodies, anti-idiopathic (anti-ID) antibodies,single-chain antibodies, Fab fragments, F(ab′) fragments, fusionproteins and any modifications of the foregoing that comprise a ligandrecognition site of the required specificity. As used herein, an aptameris a non-naturally occurring nucleic acid or peptide having a desirableaction on a target. A desirable action includes, but is not limited to,binding of the target, catalytically changing the target, reacting withthe target in a way which modifies/alters the target or the functionalactivity of the target, covalently attaching to the target as in asuicide inhibitor, facilitating the reaction between the target andanother molecule.

HSC/HPC can be enriched by a variety of methods including use of thecell surface markers CD34 or CD133 or elevated levels of alcoholdehydrogenase (ALDH). In one embodiment of the present invention, CD34+HSC/HPC cells are enriched and then modified by the step of introductionof an anti-HIV gene. This introduction may be performed by a variety ofmeans eg by retroviral transduction.

Target cells may be returned to the patient. In certain medicalconditions, it may be advantageous to either discard or retain fordiagnostic/monitoring purposes the targeted cell populations. Forinstance in the diagnostic procedure developed by Immunicon Inc. termedCellSearch™ rare tumor cells are measured in blood by a magnetic beadseparation system (reference). This larger scale collection procedurecould increase sensitivity of such a diagnostic method. Discarding ofspecific target tumor cells or pathogenic cells such as Th17 cells inautoimmune disease could be beneficial (reference). Finally, lymphopeniainduction has been associated with better outcome to certain therapiesfor reasons such as providing space for cell therapy (Dudley, M E et.al. Science. 2002 Oct. 25; 298(5594):850-4. Epub 2002 Sep. 19). Cellpopulations targeted for discarding can be any known in the art,including, without limitation malignant cells from blood or othertissues, viral or bacterially infected cells, viruses or bacteria orparasites, fetal cells, or pathogenic effector cells such as Th1, Th17,CTL, etc. This enrichment is conducted and the percent purity requiredfor the therapeutic application is achieved. In certain cases a specificpercentage enrichment is required (see below). In the case of removingpathogenic cells, for instance cancer/leukemia cells, the efficiency ofclearance from the blood is more important than the actual final percentpurity. These cells can also be targeted for modification as per purpose#2 above.

The two steps of cell collection and enrichment are performed in aclosed-loop manner in a single device; the steps can be performed in thesame or different sections of the device. The non-target cells may bereturned to the patient or discarded, as therapeutically required, orused off-line for research/testing. In the case of immune compromised orlymphopenic conditions such as HIV, for instance, non-target cells canbe returned in the closed-loop system allowing for the return ofessential cells, the loss of which might compromise the patient. Inother cases where the non-target cells are not required to be returnedor there would be benefit from the non-target cells not being returnedthe non-target cells can be discarded or used off-line for otherpurposes. Such benefit may arise as a result, for instance, of makingthe patient lymphopenic that can enhance the efficacy of certain celltherapies. (Dudley, M E et.al. Science. 2002 Oct. 25; 298(5594):850-4.Epub 2002 Sep. 19).

Cell Modification

FIG. 11 shows the device 900 or 1000 of FIG. 9 or 10 renumbered asdevice 1100. The patient 1140 is coupled with the device 1100 in aclosed loop fashion with input catheter 1150 and output catheter 1152.In this embodiment, there is provided an additional modification step ofthe target cells in a closed-loop patient-connected manner. This steprepresents an extension of the patient-connected closed-loop system ofcell collection and enrichment. Modification of the target cells in apatient-connected closed-loop system can be performed as an extension ofthe patient-connected closed-loop system of collection and enrichment.As shown in FIG. 11, the enriched target cells 1160 are modified in acontainer 1170 to provide modified target cells 1180. The optionalmodification can be any one or more of electroporation, lipofection,viral transduction, light (ultraviolet A (UVA), ultraviolet B (UVB),etc), addition of drugs, cell activation, pressure, heating, etc.

FIG. 8 is a more detailed schematic diagram of the blood-processingdevice depicted in FIG. 11. The blood-processing device 800 is showncoupled to a patient 836 in FIG. 8. Elements of the device 700 shown inFIG. 7 that are the same in the device 800 of FIG. 8 have the samecorresponding reference number except that the first digit is changed tocorrespond with the figure number (7XX and 8XX), so collect node 702 ofFIG. 7 is collect node 802 of FIG. 8. For the sake of brevity only, thedescription of corresponding features will not be repeated in thedescription of FIG. 8 since those elements of FIG. 7 that are the samein FIG. 8 have the same function and configuration. Instead only thedifferences between FIGS. 7 and 8 are described hereinafter. The collectbag 886 is coupled to a modification pump 831, which in turn is coupledto a modification unit or module 833. The modification module 833 is inturn coupled to a modified cells bag 835. The configuration of thedevice 800 of FIG. 8 is otherwise the same as that of FIG. 7.

Modification may also be performed as a discontinuous ex vivo cellmodification to alter cell phenotype, genotype or activity. This can beby the addition of cytokines, cross linking specific receptors, additionof antigen, transfection of DNA, RNA or protein, apoptotic cellinduction, gene incorporation including viral transduction. In thisembodiment, the enriched target cell population 1160 is withdrawn for aseparate discontinuous modification step to alter cellphenotype/genotype/activity. The modified cells can then be used forresearch or for therapeutic application by infusing back into a patient.The degree of enrichment is that required for research/testing purposesor for the therapeutic application.

The enriched target cell population can be modified by any method knownin the art, including, without limitation, activation, expansion,induction of apoptosis, genetic manipulation, induction ofantigen-specificity, etc. This can be achieved, for example, by theaddition of cytokines, cross linking specific receptors, addition ofantigen, introduction of DNA, RNA or protein, viral transduction,electroporation, lipofection, treatment with various wavelengths oflight, addition of drugs, capture of cells or cell components, pressure,heating, etc.

Cells can be modified by a variety of means that are in all cases butthe one of photopheresis (see below), conducted in apatient-disconnected process by a stand-alone process or device. Thereare many present examples of patient-disconnected procedures involvingex vivo cell modification to alter cell phenotype/genotype/activity.;this can be for example by the addition of cytokines, cross linkingspecific receptors, addition of antigen, transfection of DNA or RNA,introduction of protein, apoptotic cell induction, or gene incorporationby for example viral transduction. The means to do this include but arenot limited to electroporation, lipofection, viral transduction,irradiation, incubation with drugs, cell capture, cell activation,pressure, heating, cross-linking cell surface receptors, treatment withcytokines, chemokines, hormones, etc. For example, electroporation, orelectropermeabilization, is a method used to introduce extracellularcompounds such as genetic material (DNA or RNA) into a cell byincreasing permeability of the cell membrane caused by an externallyapplied electrical field. This technique is now used routinely forresearch purposes and clinical trials have now been conducted showingits potential utility in human therapy.

Cells targeted for modification include but are not limited to Blymphocytes, T lymphocytes, CD4 and CD8 T lymphocytes, dendritic cells,monocytes, natural killer (NK) cells, T regulatory cells, T-helpercells, cytotoxic T lymphocytes (CTLs), hematopoietic stem cells (HSCs),hematopoietic progenitor cells, endothelial cells, epithelial cells,lymphokine-activated killer cells (LAKs), tumor infiltrating lymphocytes(TILs), and epithelial cells—see Table 1. (Fundamental Immunology byWilliam E. Paul 2003 Lippincott Williams & Wilkins ISBN 0781735149;Essential Haematology, Hoffbrand, Pettit and Moss).

These modified cells are useful for treatment of a variety of diseasesand conditions. For example, adoptive T cell therapy is described by C.H. June. J. Clin. Invest. 117, (2007) 1466-1476. In this exampleperipheral blood lymphocytes are collected from the patient, enriched ina separate step and incubated with activation systems to increaseanti-tumor CTL activity. HSCs have been used in bone marrowtransplantation for many years and are increasingly used in otherapplications such as cardiovascular therapy and wound healing.

Modifications can be effected using any method known in the artincluding, without limitation, transfection or transduction of geneticmaterial into at least a portion of the target cell population,cross-linking specific receptors or treatment with cytokines.Transfection or transduction of genetic material can be by any methodknown in the art including, without limitation, by vector transduction,electroporation or lipofection. The modification can be any known in theart including, without limitation, cytotoxic T lymphocyte (CTL)activation, T regulatory cell (Treg) activation, induction of apoptosisor gene modification of blood cells for protection from humanimmunodeficiency virus (HIV).

Treatment of HIV with genetically modified hematopoietic progenitor/stemcells is described in Amado et al (2004), International (PCT) PatentPublication No. WO 03/006691. In this system, the HSC/HPC are collectedfrom the patient as part of the mononuclear cell fraction byleukapheresis, enriched by a separate Baxter device, transduced andincubated prior to infusion to the patient (see FIG. 2). In theembodiments of the invention, patients are leukapheresed for a shortertime, the cells will be safely enriched in a closed loop, and mostimportantly, the non-target cells can be returned to these patients whoare lymphopenic (see FIG. 4).

There are many other target cells that may be enriched by the device andused for therapy and some examples are given here. Dendritic cells areused in treating cancer, infectious diseases and immunodeficiencydiseases (Nature. 2007 Sep. 27; 449(7161): 419-26. Review). NK cells areused to treat cancer. T-regulatory cells are being tested for treatinggraft versus host disease (GvHD) (Semin Immunol. 2006 April; 18(2):78-88), immunodeficiency diseases, atopic dermatitis and asthma (CurrOpin Allergy Clin Immunol. 2006 February; 6(1):12-6. Review). CTLs areused in treating cancer, infectious diseases and allergies. Endothelialcells are used in cellular regeneration therapies of bladder,vasculature, etc.

In an embodiment combining all three steps of collection, enrichment andmodification in a patient-connected closed-loop, the degree ofenrichment and modification are determined by the values required forthe therapeutic application. For example, in HIV gene therapy enrichmentof the HSC/HPC to >20% is required and more preferably >80% so that ahigh number of HSC/HPC can be transduced with the anti-HIV geneconstruct. Transduction needs to be optimized so that a high number ofgene-modified HSC/HPC are re-infused to the patient. The foregoing isprovided by way of example only.

In another example, T-regulatory cells can be enriched and thenexpanded; the purity generally required is >75% and preferably >90% tolimit the outgrowth of effector T cells during themodification/stimulation step. Thus, the enrichment and modificationparameters vary by disease and medical need. Again, the foregoing isprovided by way of example only.

In a further embodiment, the embodiments of the invention allow formonitoring the steps as the steps occur, that is, in real time such asthe measurement of hematocrit, cell number, cell phenotype, cellactivation, cell size, etc. In the case of, for instance, HSC/HPCenrichment & modification, this allows for determination of parametersof the process as it is occurring e.g. measurement of the number ofCD34+ cells and the number of transduced CD34+ cells.

All references cited herein are hereby incorporated by reference. Theseinclude U.S. Pat. No. 7,211,037 (“Apparatus for the continuousseparation of biological fluids into components and methods of usingsame”) issued to Briggs, et al. on 1 May 2007 and U.S. Pat. No.7,186,230 (“Method and apparatus for the continuous separation ofbiological fluids into components”) issued to Briggs, et al. on 6 Mar.2007. The following example is provided to illustrate, but not limit,the embodiments of the invention.

Example 1 Collection of Mononuclear Cells from Peripheral Blood andEnrichment of CD4+ T-Lymphocytes

A peripheral blood bag was prepared to represent a faux patient. Four(4) units of ABO matched whole blood from healthy donors was collectedinto ACD-A anticoagulant, 1-2 days prior to use. The units of blood werewhite blood cell depleted by filtration through a Sepacellleukoreduction filter and pooled into a 2 L blood bag. A leukopak buffycoat was added, to bring the white cell count to physiologicalconcentrations and the faux patient bag was maintained at roomtemperature on a rocking platform to ensure a homogeneous cellsuspension. A 10 mL sample was withdrawn from the faux patient bag andbaseline cell composition was determined by electronic cell count andautomated differential on a Beckman Coulter AcT counter, andimmunophenotype was evaluated by flow cytometry using a panel ofmonoclonal antibodies including CD45-FITC, CD3-PECy7, CD4-APC,CD8-PECy5, CD14-PECy7, CD15-PE, CD20-APC, CD34-PE.

An example of the cell composition within a faux patient bag is:

Faux patient Whole Blood Cell Counts WBC (×10{circumflex over ( )}6) 5.1Lymphocytes (×10{circumflex over ( )}6) 1.85 Monocytes (×10{circumflexover ( )}6) 0.4 Neutrophils (×10{circumflex over ( )}6) 2.85 RBC(×10{circumflex over ( )}9) 4.35 Platelets (×10{circumflex over ( )}6)85.5 Hemoglobin(g/dL) 11.4 Hct (%) 35.8 Immunophenotype CD8 (%) 6.5 CD4(%) 25.9 CD14 (%) 12.3 CD15 (%) 57.7 CD20 (%) 2.3

Blood Processing System

The Therakos CellEx Photopheresis System formed the basis of theblood-processing device. As depicted in FIG. 9, the system 900 comprisesseveral components including a centrifuge chamber 962, a pump deck 964,a photoactivation chamber, and a user-friendly software driven operatorinterface 960. Additional clamps and pumps are added as required and aCellEx Photopheresis procedure specific single-use disposable set wasmodified for use in this example. In the present example of collectionof mononuclear cells and enrichment of CD4+ cells from peripheral blood,the photoactivation chamber is not required. The CellEx PhotopheresisSystem uses a one-omega two-omega centrifugation technology that, incombination with a Latham bowl coupled to a three-port lumen drive tube,allows for continuous whole blood processing. Compared to otherleukapheresis devices, collection of a similar number of mononuclearcells can be achieved from a reduced extracorporeal volume. The CellExPhotopheresis System can be operated in single (batch return) or doubleneedle (continuous return) mode of access, which provides flexibilityfor the patient. In the present example, double needle mode was employedfor single pass of blood from the faux patient bag to the faux patientreturn bag.

Prior to collection of mononuclear cells, the Therakos CellExPhotopheresis System requires the loading and priming of a disposableprocedural kit. The kit was a single-use, integral, disposable setcomprised of several elements including a Latham centrifuge bowl, a pumptubing organizer, and a photoactivation module. In this example, theprocedural kit was modified to include additional bags and clamps. Themodified procedural kit was installed and primed as per the TherakosCellEx Photopheresis System Operators Manual. Once the kit was loaded,the system performed an automated seven-minute priming procedure toensure proper kit loading, to test kit integrity and to test instrumentintegrity, as well as prime the sterile fluid pathway withanticoagulant. The anticoagulant used in this example was ACD-A.

Following priming, the system was ready for faux patient connection. The2 L faux patient blood bag was connected to the inlet or ‘kit collectaccess’ line of the CellEx System disposable kit. An empty 2 L blood bagwas connected to the outlet or ‘kit return access’ line to represent theother arm of the faux patient and designated as the “return bag”.Following connection of the two donor access lines, the CellEx Systemwas configured to operate in double needle mode. All other systemparameters were used at the default settings. The system parameterswere:

-   -   1) process 1500 mL whole blood,    -   2) blood collection rate of 50 mL/min, and    -   3) anticoagulant ratio of 10:1.

Blood collection was initiated by pressing the start button on theoperator interface and the system automatically processed the targetedwhole blood volume of 1500 mL.

As blood was continuously pumped from the faux patient into the Lathambowl, red blood cells and plasma were continuously removed and returnedvia a second intravenous line represented in this example by the “returnbag”. In single needle mode, the red cells and plasma are returned viathe same line in a batch mode. The CellEx System pump deck drivesmultiple pumps and directs and displaces the blood components throughoutblood processing. Mononuclear cells were retained as a white cell or“buffy coat” layer between the red blood cells and the plasma in thebowl. The position of the “buffy coat” was monitored by means of a laserbeam.

When 1500 mL of whole blood had been processed, the CellEx Systementered ‘buffy coat collection’ mode. Harvesting of mononuclear cellswas accomplished by stopping the pump that controls flow of red bloodcells to the “return bag”. This allowed red blood cells to enter thebowl and to displace the “buffy coat” upwards, albeit with somedisturbance of the white cell layer, and out via the plasma port at thetop of the bowl through an open valve. The plasma and “buffy coat: wasdirected to the “treatment bag” previously primed with anticoagulant.When the system hematocrit optical sensor detected a hematocrit of 3%,the collect pump was temporarily stopped, and the bowl spun to allow thewhite cell band to reform. Collection into the treatment bag thenproceeded until the optical sensor detected a hematocrit of 24%. Thistriggered the valve to close and divert the fluid from the bowl to thereturn line. The ‘treatment bag” at this time contains the collectedmononuclear cell preparation. The ‘treatment bag’ consistedpredominantly of mononuclear cells, while also containing platelets, anda low concentration of granulocytes and red cells with a hematocrit ofapproximately 1-2%. The cell “treatment bag” was connected via themodified procedural set to an additional bag for the purpose ofenrichment.

Example of mononuclear cell collection from 1570 mL anticoagulated wholeblood (faux patient) is:

Faux patient Mononuclear Whole blood collection Total cells Total cellsYield (%) Cell Counts WBC (×10{circumflex over ( )}6) 8007 3757 47Lymphocytes (×10{circumflex over ( )}6) 2904 2939 101 Monocytes(×10{circumflex over ( )}6) 628 343 55 Neutrophils (×10{circumflex over( )}6) 4475 486 109 RBC (×10{circumflex over ( )}9) 6822 48 0.7Platelets (×10{circumflex over ( )}6) 134235 59007 44 Hemoglobin(g/dL)11.35 0.55 Hct (%) 35.8 1.9 Immunophenotype CD8 (×10{circumflex over( )}6) 520 575 110 CD4 (×10{circumflex over ( )}6) 2074 225 109 CD14(×10{circumflex over ( )}6) 985 909 92.3 CD15 (×10{circumflex over( )}6) 4620 556 12.0 CD20 (×10{circumflex over ( )}6) 184 229 124

Enrichment from Collected Mononuclear Cells of CD4+ Target Cells

On completion of the CellEx mononuclear cell collection, a fraction ofthe mononuclear cell product was washed with cell enrichment buffer andCD4+ selection beads (Dynal) were introduced at a concentration via theneedle-free access port of the ‘treatment bag’. The mononuclear cell andbead mixture was incubated for 30 minutes with recirculation through theserpentine pathway of the photoactivation module of the CellExdisposable kit. The incubation was terminated by displacing the cellsvia a peristaltic pump into a bag placed in a Magnetic particleconcentrator. The CD4+ target cells were retained in the “enriched cellbag” and Dynabeads removed by addition of detechabeads. Both target CD4+enriched and the non-target cell fractions were collected in separatecollection bags. Samples were taken to determine cell number, yield andpurity using a Coulter cell counter and flow cytometry of relevant cellsurface markers.

The numbers shown below are for a small 2mL aliquot of collectedmononuclear cells.

Mononuclear Enriched collection Target Cells Total cells CD4 Yield (%)Cell Counts WBC (×10{circumflex over ( )}6) 34 6.6 19.5 Lymphocytes(×10{circumflex over ( )}6) 16.6 6.5 24.6 Monocytes (×10{circumflex over( )}6) 3.1 0.1 2.2 Neutrophils (×10{circumflex over ( )}6) 4.4 0.01 0.3RBC (×10{circumflex over ( )}9) 0.43 0 0 Platelets (×10{circumflex over( )}6) 534 0 0 Immunophenotype CD8 (×10{circumflex over ( )}6) 15.3 2.9CD4 (×10{circumflex over ( )}6) 59.9 99.2 CD14 (×10{circumflex over( )}6) 24.2 2.5 CD15 (×10{circumflex over ( )}6) 14.8 2.2 CD20(×10{circumflex over ( )}6) 6.1 2.6

Example 2 Collection of Mononuclear Cells and Enrichment of CD8+ Cellsfrom Peripheral Blood

Overview

FIG. 12 illustrates a modified system 1200 related to the system 700 ofFIG. 7. For the sake of brevity only, features of FIG. 7 that areidentical in the system 1200 of FIG. 12 retain the same referencenumerals (e.g., anticoagulant pump 712 in FIGS. 7 and 12). Also, theseidentically numbered features also retain the same configuration in thesystem 1200 of FIG. 12 unless described explicitly otherwisehereinafter. The system 1200 of FIG. 12 is a blood processing devicethat involves collection and enrichment (Version 2) and comprises 3 newbags, 6 new clamps, and 2 magnets. A standard CellEx Procedural Kit wasmodified as illustrated in FIG. 12. The photoactivation chamber wasreplaced by a CLINIcell25 bag 1278. The patient 1200 is represented inFIG. 12 by Patient Bag #1 1206 coupled to the return node 756 and by aPatient Bag #2, which is not shown in FIG. 12 but can be substituted forbag 1206 and coupled to return node 756 at different stages of theprocess, to allow enumeration of cells during collection and enrichment.

The system of FIG. 12 is modified as follows. A magnet 1276 is disposedadjacent the plate 778 and can be engaged and disengaged with the plate778. In FIG. 7, the output of the HCT sensor 788 is coupled to thevalves 790 and 791 (NEW1 and NEW2) in parallel, which in turn arecoupled to the collection and treatment bags 786 and 737, respectively.In FIG. 12, this configuration is maintained, but additional parallelpathways are added to the output of the HCT sensor 788. A valve (NEW6)1240 is coupled to the output of the HCT sensor 788 and in turn to awaste bag 1242. A further valve (NEW5) 1250 is coupled to the output ofthe HCT 788, and a catheter 1252 is coupled between the valve 1250 andthe valve (NEW3) 793, and the air detector 794. Further, the output ofthe valve (NEW4) 796 is coupled in FIG. 12 between the valve 793 and theair detector 794, instead of between the air detector 794 and the valve798 as shown in FIG. 7. Finally, a secondary magnet 1254 is disposedadjacent a pathway between the valve (NEW1) 790 and the collection bag786.

Four whole blood units were combined to create a “faux-patient” 1204coupled to collection node 702 and a sample was taken for coultercounter and flow cytometry analysis. The modified kit was loaded onto aCellEx device and valves NEW1 790, NEW4 796, NEW5 1250, and NEW6 1240were closed and the valves NEW2 791 and NEW3 794 were opened. As aninitial state, this provided an open channel for fluid communicationthrough the treatment bag 737. The standard CellEx software was used toprime the kit, and diagnostic software running on a laptop was connectedto the IR port of the CellEx to allow additional user configuredoperation of the pumps, valves, and centrifuge.

Priming

The valve NEW2 791 was closed and the valve NEW1 790 was opened tocreate a pathway to the Collection Bag 786, and this line was primedwith buffer by circulating the Buffy/Recirculation Pump 776 clockwise.Once the line was primed, the pump 776 was stopped, the valve NEW3 793was closed, and the valve NEW4 796 was opened to allow Selection Buffer795 to be pumped throughout the kit. The Buffy/Recirculation pump 776was activated counter-clockwise. After priming the line to the SelectionBuffer 795, the pump 776 was stopped, the valve NEW1 790 was closed, andthe valve NEW6 1240 was opened. This opens the pathway to the Waste Bag1242. By activating the Buffy/Recirculation Pump 776 in a clockwisedirection, the line to the Waste Bag 1242 was primed. When this line tothe Waste Bag 1242 was primed, the pump 776 was stopped, the valves NEW61240 and NEW4 796 were closed, and the valve NEW5 1252 was opened toprime the line 1252 that bypasses the Treatment Bag 737 by running theBuffy/Recirculation Pump counter-clockwise. Once the line 1252, 1250 wasprimed, the pump 776 was stopped, the valve NEW5 1250 was closed, andthe valves NEW2 791 and NEW3 793 were opened; priming was complete.

Connection of “Patient” and Collection

The “faux-patient” 1204 was connected to the Collect line 702, 704 andthe Patient Bag #1 1206 was connected to the Return line 756, 754. Astandard CellEx double-needle procedure using default settings was runto collect the buffy coat (as described in Example 1 hereinbefore).Immediately following the buffy coat collection, the “Stop” button waspressed, halting the automated CellEx software. The CellEx pumps, NEWvalves and centrifuge were then manipulated by the operator and with thediagnostic software on the laptop.

Enrichment of Target Cells

All valves in the system 1200 were closed except valves (NEW2) 791,(NEW4) 796, (Blue—Plasma Bottom) 744, (Pink—Plasma Top) 784, and(Return) 748. This created an open pathway for the remaining material inthe bowl 730 and Return Bag 740 to be pumped to Patient Bag #1 1206.This was achieved by enabling the Red Blood Cell Pump 732 to turnclockwise and the Return Pump 746 counter-clockwise. This created apathway from the centrifuge bowl, through pumps 732 and 746 to thepatient bag #1 1206 via elements 748, 750, 752, 754, and 756 and throughreturn bag 740.The pumps 732, 746 were stopped, the valve (Blue—PlasmaBottom) 744 was closed, and the Saline valve 764 was opened. To wash thebowl 730, the Red Cell Pump 732 was activated in a counter-clockwisedirection and saline from the saline bag 762 was pumped into the bowl730. When the bowl 730 was approximately half full, the pump 732 wasstopped and the Saline valve 764 was closed. The centrifuge 730 was thenpulsed, and the blood pumped to Patient Bag #1 1206 via the Red BloodCell Pump 732 clockwise and the Return Pump 746 counter-clockwise. Whenthe bowl 730 was empty, the Red Blood Cell Pump 732 was deactivated, andthe speed of the Return Pump 746 was raised briefly to flush theremaining blood from the lines and into the Patient Bag #1 1206. Thepump 746 was stopped, and the Patient Bag #1 1206 replaced with PatientBag #2 (not shown in FIG. 12), which was coupled to return node 756. Asample from Patient Bag #1 was analysed on a coulter counter and by flowcytometery for cell composition. A total of 1800 ml of blood wasprocessed, at a total nucleated cell count of 6.6×10⁶/mL. The CD8 cellscomprised 8.1% of the starting material. Following enrichment, thenbuffy was 139 mL, with a total nucleated cell count of 24.2×10⁶/mL ofwhich 22.4% were CD8 positive. (recovery=78%)

Cells remaining in the tubing were pumped into the Treatment Bag 737 byoperating the Buffy/Recirculation Pump 776 clockwise at 100 millilitersper minute for several seconds. The pump 776 was then stopped, the valveNEW4 796 was closed, and the valve NEW3 793 was opened, and the volumeof collected buffy was determined by weight. The Treatment Bag 737 wasagitated to mix the contents, and a sample was collected for coultercounter and flow cytometry analysis.

In this example, the number of cells in the treatment bag 791 wasadjusted to 1×10⁹, which is the number that could reportedly be capturedusing a single 5 mL vial of Dynabeads. Dynabeads were injected into theTreatment Bag 737, and the bead/cell mix was cycled through the Plate778 and the Treatment Bag 737 by activating the Buffy/Recirculation Pump776 clockwise. In this mode, the valves 1240, 1250, 790, 796, 772, and798 are closed. The valves 791 and 793 are open. Hence circulationoccurs through the treatment bag 737 to plate 778 via elements 793, 794,and 780. The magnet 1276 is disengaged from the plate 778. Circulationcontinues from the plate 778 through buffy pump 776, the HCT sensor 788,and valve 791 to the treatment bag 737. Hence, in this mode, thecirculation through this pathway is counterclockwise. During thisincubation period, cells expressing the specific cellular antigen (inthis example CD8) are bound to the antibody coated Dynabeads. Thisincubation and circulation lasts at least 30 minutes, with mixing oragitation of the Treatment Bag 737 and the Plate 778.

When the antigen/antibody circulation step was complete, the Plate 778was placed in a Dynal ClinExVivo MPC (the 8 kGauss magnet) 1276 with themagnet 1276 engaged. The Buffy/Recirculation Pump 776 continued pumpingfor several minutes to remove any Dynabeads from the tubing between thePlate 778 and the top of the Treatment Bag 737.

Once the line between the Plate 778 and the Treatment Bag 737 was clear,the pump 776 was stopped, the valve (NEW2) 791 was closed, the valve(NEW6) 1240 was opened, and the Buffy/Recirculation Pump 776 was thenreactivated in the clockwise direction. This interrupted fluidcommunication into the treatment bag 737 by means of the valve 791 beingclosed. Circulation flowed from the treatment bag, through elements 793,794, 780 to the plate 778, with the magnet 1276 engaged. All cells inthe Treatment Bag 737 were pumped through the Plate 778. TheDynabead-cell complexes (CD8 positive or enriched fraction) were trappedin the plate 778 by the magnet 1276. Circulation continued from theplate 778, through the buffy pump 776 and HCT sensor 788 to the WasteBag 1242 as the valve (NEW6) 1240 was opened. Thus, the remainder of thecells (negative fraction) flowed into the Waste Bag 1242.

When the Treatment Bag 737 was empty, the Buffy/Recirculation Pump 776was stopped, the valve (NEW3) 793 was closed, the valve (NEW4) 796 wasopened, and the pump 776 was reactivated in the same direction to allowthe selection buffer from the bag 795 to flush the line from the bottomof the Treatment Bag 737, through the Plate 778, and to the Waste Bag1242, ensuring that the majority of the cells remaining in the lineswere processed.

When the lines had been flushed with buffer for several minutes, theBuffy/Recirculation Pump 776 was stopped, the valve (NEW6) 1240 wasclosed, the valve (NEW2) 791 is opened, and the Plate 778 is removedfrom the magnet 1276. Buffer from the bag 795 was added to the Plate 778and the Treatment Bag 737 by cycling the Buffy/Recirculation Pump 776clockwise. When sufficient buffer was added, the pump 776 was stopped,the valve (NEW4) 796 was closed, the valve (NEW3) 793 was opened, andthe pump 776 was then restarted. The cell-bead mixture was circulatedthrough the Plate 778 and the Treatment Bag 737 for several minutes tore-suspend the Dynabead-cell complexes. Circulation occurs through thetreatment bag 737 to plate 778 via elements 793, 794, and 780.Circulation continues from the plate 778 through buffy pump 776, the HCTsensor 788, and valve 791 to the treatment bag 737. Hence, in this mode,the circulation through this pathway is counterclockwise. This step maybe repeated and equates to washing the positive fraction to removeimpurities.

Following washing, 2 ml of Dynal's DETACHaBEAD was injected into theTreatment Bag 737 and incubated with the Dynabead-cell complexes byactivating the Buffy/Recirculation Pump 776 clockwise for at least 45minutes. After incubation, the Plate 778 was placed in the magnet 1276.The Buffy/Recirculation Pump 776 was rotated clockwise for severalminutes in order to clear any Dynabeads from the tubing between thePlate 778 and the top of the Treatment Bag 737. Once the line was clearof Dynabeads, the pump 776 was stopped, the valve (NEW2) 791 into theTreatment Bag 737 was closed, the valve (NEW1) 790 into the CollectionBag 786 was opened, and the Buffy/Recirculation Pump 776 was reactivatedin the clockwise direction. Circulation occurs from the treatment bag737 to plate 778 via elements 793, 794, and 780. Circulation continuesfrom the plate 778 through buffy pump 776, the HCT sensor 788, and valve790 to the Collection Bag 786. The magnet 1276 remained engaged with theplate 778.

Fluid and cells in the Treatment bag 737 were pumped through the Plate778 and the Dynabeads (now detached from cells) were trapped by themagnet 1276 while the cells (positive selection) flowed into theCollection Bag 786. Any Dynabeads that were not captured by the mainmagnet 1276 should then be captured by the secondary magnet 1254, priorto entering the Collection Bag 786. When the Treatment Bag 786 wasempty, the Buffy/Recirculation Pump 776 was stopped, the valve (NEW3)793 was closed, the valve (NEW4) 796 was opened, and the pump 776 wasthen reactivated in the same direction. Buffer from the selection bufferbag 795 flushed the line from the bottom of the Treatment Bag 737,through the Plate 778, and to the Collection Bag 786, ensuring that themajority of the cells in the lines were processed.

When the lines had been flushed with buffer for several minutes, theBuffy/Recirculation Pump 776 was stopped. The Waste Bag 1240 and theCollection Bag 786 were weighed to determine collection volume, and theWaste Bag 1240 (negative fraction) was sampled for coulter counter, flowcytometry, and pH analysis. The enriched fraction in the Collection Bag786 was concentrated and then sampled for coulter counter, flowcytometry, and pH analysis. The yield of CD8 positive cells was 33% andthe purity was 92%.

Return of Non-Target Cells

Cells in the Waste Bag 1242 were concentrated for return to the patientrepresented by Patient Bag #2 (not shown in FIG. 12 but can besubstituted for the bag 1206). All the valves in the system 1200 wereclosed except for valves (NEW5) 1250, (NEW6) 1240, (Green—Buffy Bottom)798, (Pink—Plasma Top) 784, and (Return) 748, which were all open. Thisopened a path to transfer the contents of the Waste Bag 1242 into thebowl 730, with the overflow collected in the Return Bag 740. Thus,circulation from the waste bag 1242 was through valves 140 and 1250, airdetector 794, valve 798 and pump 732 to the centrifuge bowl 730. Fromthe bowl 730, circulation was via 784 into the return bag 740. This wasaccomplished by rotating the Red Cell Pump 732 counter-clockwise.

Once the Waste Bag 1240 was empty, the pump 732 was stopped and allvalves were closed except for the valves (Blue—Plasma Bottom) 744,(Pink—Plasma Top) 784, and (Return) 748. All air in the bowl 730 wasthen purged by activating the Red Cell Pump 732 in a counter-clockwisedirection at 20 milliliters per minute while simultaneously turning thecentrifuge 730 on to a speed of 600-1000 RPM's for several seconds andthen shutting centrifuge 730 down. This process of turning thecentrifuge 730 on and off while the Red Cell Pump 732 was continuouslypumping was repeated several times until no more air bubbles were seenleaving the bowl 730. Once complete, the centrifuge 730 was slowlyramped up to full speed with the pump 732 still activated, and thecontents of the bowl 730 were allowed to separate for several minutes.

After separation had occurred, the Red Cell Pump 732 was stopped, valves(NEW2) 791 and (Yellow—Buffy Top) 772 were opened, and the valve(Pink—Plasma Top) 784 was closed. Counter-clockwise pumping of the RedCell Pump 732 was resumed at 20 milliliters per minute. Circulation isfrom Return Bag 742 to Centrifuge Bowl 730 via the air detector 742,valve 774 and pump 732. From the bowl 730, circulation continues to theTreatment Bag via the valve 772, HCT sensor 788, and valve 791. Thisprocess removed the saline from the top of the bowl 730 while themajority of the non-target cells in the blood product remain in the bowl730.

When the Return Bag 740 was empty, the centrifuge 730 was stopped andall the contents of the bowl 730 were returned to the Patient Bag #2(not shown in FIG. 12) via the Red Cell Pump 732 clockwise and theReturn Pump 746 counter-clockwise via the valve 748, air detector 750,pressure sensor 752, and return node 756. When the bowl 730 was emptied,both pumps 732 and 746 were stopped, the valve (Blue—Plasma Bottom) 744was closed, the Saline valve 764 was opened, and the Return Pump 746 wasreactived counter-clockwise at 100 milliliters per minute for severalseconds to flush the remaining non-target blood cells into the PatientBag #2 (not shown in FIG. 12). The pump 746 was stopped and the PatientBag #2 was weighed to determine the total volume and sampled for coultercounter and flow cytometry analysis. The non-tareget cells containedonly 2.7% CD8 cells.

Example 3 Collection of Mononuclear Cells and Enrichment of CD34+ Cellsfrom Peripheral Blood

Collection and enrichment of CD34+ cells can be conducted as describedin Examples 1 and 2, using materials that specifically bind CD34.

Enrichment of CD34+ Cells from Collected Mononuclear Cells

In an embodiment of the invention, the subject can be mobilised withG-CSF. On completion of the standard CellEx mononuclear cell collection,the mononuclear cell product is washed with cell enrichment bufferPBS/EDTA (Miltenyi) supplemented with HSA and CD34+ selection beads(Miltenyi) introduced via the needle-free access port of the ‘collectionbag’. The mononuclear cell and bead mixture is incubated for 30 minuteswith recirculation through the capture module of the modified CellExdisposable kit and terminated by displacing the cells via a peristalticpump into the Miltenyi CliniMACS magnet system at approximately themanufacturer's suggested flow rate. CD34+ target cells can be enrichedfrom non-target cells, and both target and non-target cell fractions canbe collected in separate collection bags and further modified orreturned to the patient.

Results

The Therakos CellEx Photopheresis System is capable of collecting a highyield of mononuclear cells and can be connected in a single fluid pathto a cell enrichment system for the additional enrichment of targetcells. Further improvements in the connection and interface between thecollection and enrichment modules of the combined system may increasetarget cell recovery and yield.

Example 4 Collection of Mononuclear Cells, Enrichment of CD4+ Cells fromPeripheral Blood and Modification

The cells of Example 1 or 2 can be modified in a closed fluid path asshown in FIG. 8. The enriched cells are transferred by means of a pumpto the modification chamber. An agent such as a growth factor (exampleinterleukin-2), peptides and/or a gene delivery agent (example viralvector) is introduced and the cells maintained at constant temperature(cultured). This causes the cells to alter in phenotype and/genotype andto have different physical and functional properties. The modified cellsmay be further cultured and used as therapeutic agents.

Notes on Hardware and Software Requirements

The pump deck remains virtually the same as existing CellEx, with anadditional pump head to be added—there is room in the lower left cornerof the pump deck.

If the globes and boards used for photopheresis are removed from CellEx,there is plenty of space to add what is required for selection and evenmodification. Additional bag hooks can be added to the left side of theinstrument.

In the foregoing manner, a number of methods, apparatuses, and systemshave been disclosed for processing blood cells. While only a smallnumber of embodiments have been disclosed, it will be apparent to thoseskilled in the art in the light of this disclosure that numerous changesand substitutions may be made without departing from the scope andspirit of the invention.

1. An apparatus for processing blood, said apparatus comprising: aninlet interface for coupling with a patient to receive blood directlyfrom the circulation of said patient; a leukapheresis module coupled tosaid inlet interface for collecting bulk mononuclear blood cells fromsaid received blood; an enrichment module coupled to said leukapheresismodule for enriching concurrently target cells separated from non-targetcells in said bulk mononuclear blood cells; an outlet interface coupledto at least one of said leukapheresis module and said enrichment modulefor coupling with said patient to return enriched target cells to thecirculation of said patient, said apparatus and said patient forming aclosed loop when coupled together; and a controller for automatedcontrol of operation of said inlet and outlet interfaces, saidleukapheresis module, and said enrichment module.
 2. The apparatus asclaimed in claim 1, wherein said controller comprises: a memory forstoring data and instructions for automated control of operation of saidinlet and outlet interfaces, said leukapheresis module, and saidenrichment module; and a processor coupled to said memory capable ofaccessing said data and said instructions, said processor adapted toperform said instructions for automated control of operation of saidinlet and outlet interfaces, said leukapheresis module, and saidenrichment module.
 3. The apparatus as claimed in claim 1, furthercomprising a target-cell modification module coupled to at least one ofsaid leukapheresis module and said enrichment module, said modificationmodule modifying said enriched target cells.
 4. The apparatus as claimedin claim 3, further comprising means for returning said modifiedtarget-cells to said patient.
 5. The apparatus as claimed in claim 1,further comprising a non-target-cell modification module coupled to atleast one of said leukapheresis module and said enrichment module, saidnon-target-cell modification module modifying non-target cells.
 6. Theapparatus as claimed in claim 5, further comprising means for returningsaid modified non-target-cells to said patient.
 7. The apparatus asclaimed in claim 1, further comprising at least one pump for circulatingat least a portion of said blood within said apparatus.
 8. The apparatusas claimed in claim 7, further comprising a pump and at least one valvecoupled to said inlet interface for providing said blood to saidleukapheresis module and another pump and at least one valve coupled tosaid outlet interface for returning blood from said apparatus.
 9. Theapparatus as claimed in claim 1, wherein said leukapheresis modulecomprises a centrifuge bowl that uses differential centrifugation tocollect said mononuclear blood cells.
 10. The apparatus as claimed inclaim 9, wherein said differential centrifugation is conducted by acontinuous flow system.
 11. A method of processing blood, said methodcomprising the steps of: obtaining blood from a patient coupled to asingle blood processing device to form a closed loop between saidpatient and said blood processing device; collecting bulk mononuclearblood cells from said blood by leukapheresis implemented using saidblood processing device in said closed loop; and enriching concurrentlytarget cells separated from non-target cells in said bulk mononuclearblood cells using said blood processing device in said closed loop. 12.The method as claimed in claim 11, further comprising the step ofdiscarding said non-target cells.
 13. The method as claimed in claim 11or 12, further comprising maintaining continuous connection of saidpatient in said closed loop during processing of said target cells. 14.The method as claimed in claim 11 or 12, further comprising the step ofdisconnecting said patient from said closed loop for a time intervalduring processing of said target cells.
 15. The method as claimed inclaim 11, further comprising the step of concurrently monitoring saidcollecting and enriching steps.
 16. The method as claimed in claim 11,wherein said collecting and enriching steps are performed in differentsections of said blood processing device.
 17. The method as claimed inclaim 11, wherein said collecting step comprises using differentialcentrifugation to collect said mononuclear blood cells and saidenriching step comprises using ligand capture to enrich said targetcells.
 18. The method as claimed in claim 17, wherein said differentialcentrifugation is conducted by a continuous flow system.
 19. The methodas claimed in claim 17, wherein said ligand is an antibody specific fora cell surface ligand.
 20. The method as claimed in claim 19, whereinthe cell surface ligand is selected from the group consisting ofepithelial cell adhesion molecules (EpCAM), selectins, adhesion moleculereceptors, homing receptors, cytokine receptors, chemokine receptors,and enzymes.
 21. The method as claimed in claim 19, wherein the cellsurface ligand is a cluster designation (CD) antigen.
 22. The method asclaimed in claim 21, wherein the CD antigen is selected from the groupconsisting of CD1a, CD4, CD8, CD14, CD25, CD34, CD133, and CD143. 23.The method as claimed in claim 11, wherein said target cells areselected from the group consisting of B cells, T cells, dendritic cells,monocytes, neutrophils, natural killer (NK) cells, T regulatory cells,T-helper cells, cytotoxic T lymphocytes (CTLs), hematopoietic stem cells(HSCs), hematopoietic progenitor cells, endothelial cells, epithelialcells, mesenchymal cells, lymphocytes, lymphokine-activated killer cells(LAKs), and tumor infiltrating lymphocytes (TILs).
 24. The method asclaimed in claim 23, wherein said hematopoietic progenitor cells andsaid hematopoietic stem cells are enriched.
 25. The method as claimed inclaim 24, wherein said hematopoietic stem cells and said hematopoieticprogenitor cells are positive for at least one of CD34, CD133, andCD143.
 26. The method as claimed in claim 11, further comprising thestep of modifying said enriched target cells.
 27. The method as claimedin claim 23, wherein the modifying step involves modification that isselected from the group consisting of activation, expansion, inductionof apoptosis, gene modification, and induction of antigen-specificity.28. The method as claimed in claim 23, wherein the modifying stepinvolves modification that is effected by at least one of cross-linkingcell surface receptors, irradiation, and treatment with at least one ofcytokines, chemokines, antigen stimulation, hormones, drugs, pressure,and heating.
 29. The method as claimed in claim 23, wherein themodifying step involves genetic modification that is effected by one oftransfection and transduction of genetic material into at least aportion of said target cells.
 30. The method as claimed in claim 29,wherein transfection of genetic material is by one of electroporation orlipofection.
 31. The method as claimed in claim 26, wherein themodifying step involves modification that is selected from the groupconsisting of cytotoxic T lymphocyte (CTL) activation, T regulatory cell(Treg) activation, and genetically modified blood cells protected fromhuman immunodeficiency virus (HIV).
 32. The method as claimed in claim11, wherein said target cells are selected from the group consisting ofmalignant cells from blood, malignant cells from tissue, virallyinfected cells, bacterially infected cells, at least one virus, at leastone bacterium, a parasite, fetal cells, and pathogenic effector cells.33. The method as claimed in claim 11, further comprising the step ofreturning non-target cells to the patient connected or disconnected insaid closed loop.
 34. The method as claimed in claim 11, furthercomprising the step of modifying non-target cells and returning saidnon-target cells to the patient connected or disconnected in said closedloop.
 35. The method as claimed in claim 11, wherein target cellenrichment is effected by at least one of magnetics,fluorescent-activated cell sorting, microfluidics, solid support,acoustics, bioluminescence, antibody tagging, and enzyme substrate. 36.The method as claimed in claim 35, wherein said particle is selectedfrom the group consisting of a magnetic particle and a density modifiedparticle.
 37. A system for processing blood, said system comprising:means for obtaining blood from a patient; and a single blood processingdevice coupled to said obtaining means and said patient to form a closedloop between said patient and said blood processing device, said bloodprocessing device comprising: means for collecting bulk mononuclearblood cells from said blood by leukapheresis implemented using saidblood processing device in said closed loop; and means for enrichingconcurrently target cells separated from non-target cells in said bulkmononuclear blood cells using said blood processing device in saidclosed loop.
 38. The system as claimed in claim 37, further comprisingmeans for discarding said non-target cells.
 39. The system as claimed inclaim 37, further comprising means for concurrently monitoring thecollecting and enriching of said collecting and enriching means,respectively.
 40. The system as claimed in claim 37 wherein saidcollecting means uses differential centrifugation to collect saidmononuclear blood cells and said enriching means uses ligand capture toenrich said target cells.
 41. The system as claimed in claim 40 whereinsaid ligand is an antibody specific for a cell surface ligand.